BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1624-1630
Vol. 189, No. 3, 1992 December 30, 1992
TSH-INDUCED GALACTOSE INCORPORATION AT THE NH2 TERMINUS OF THYROGLOBULIN SECRETED BY FRTL-5 CELLS Bruno Di Jesol and Fabrizio Gentile Centro di Endocrinologia e Oncologia Sperimentale de1 C.N.R. and Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples 2nd Medical School, Via Pansini, 5, 80131 Naples, Italy Received
November
20,
1992
SUMMARY : FRTL-5 cells were cultured in media containing standard growth factors with or without TSH, plus labeled precursors of N-linked secreted in the medium was oligosaccharide chains. The thyroglobulin purified and fragmented with CNBr. Three peptides were identified by NH2-terminal sequencing, that were labeled mainly with D-[2-3H]mannose, independent of TSH. One of them, corresponding to the NHz-terminus of thyroglobulin, incorporated both more D-[2-3Hlmannose and more D-[ l3Hlgalactose upon TSH addition. These data likely reflect a TSH-induced increment of N-linked glycosylation at the NH2-terminus of thyroglobulin, 0 1992 mostly with the maturation of high-mannose to complex chains. Rcademlc mes*,Inc.
Thyroglobulin is the dimeric glycoprotein of 660 kDa providing the backbone for the synthesis of thyroid hormones. Carbohydrates compose 10% of its mass; major oligosaccharide chains of human and bovine Tg include N-linked chains of high-mannose type, consisting of mannose and and complex type, containing in addition galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid and sulfate groups [ 1, 21. O-linked chains of human Tg include galactosamine-containing chains and chondroitin 6sulfate-like chains, containing N-acetylgalactosamine and glucuronic acid
1 To whom correspondence should be addressed. Abbreviations: Tg, thyroglobulin; rTg, rat thyroglobulin; bTg, bovine hTg, human thyroglobulin; [sHIman, D-[2-3H] mannose; thyroglobulin; [3H]gal, D-[ 1-sH]galactose; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 0006-291X/92
$4.00
Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1624
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
[2-41. TSH promotes the sialylation of the oligosaccharide chains of Tg secreted by pig thyroid cells in primary culture [5] and the maturation of the high-mannose to complex chains of the Tg secreted by FRTL-5 cells [6]. This study was undertaken in order to determine: 1) the kinds of Nlinked oligosaccharide chains present in defined regions of the sequence of Tg secreted by FRTL-5 cells; 2) the location of the oligosaccharide chains maturing from the high-mannose to the complex type under the effect of TSH. The Tg secreted by FRTL-5 cells, cultured both in the absence and in the presence of TSH, plus labeled precursors of oligosaccharide chains (D[2-JH]mannose for high-mannose chains and D-[l-3Hlgalactose for complex chains), was purified and fragmented with CNBr. The peptides produced were isolated and identified by NHz-terminal sequencing and alignment with the gene- and cDNA-derived sequences of rTg, bTg and hTg [7-101. MATERIALS
AND METHODS
Materials: D-[2-3Hlmannose (10 Ci/mmol) and D-[ 1-3Hlgalactose (5 Ci/mmol) from Amersham, hormones from Calbiochem (Insulin) or Sigma. Cell culture: FRTL-5 cells (ATCC CRL 8305) were routinely cultured in Coon’s modified Ham’s F12 medium plus 5% calf serum, glycyl-histidyllysine (10 rig/ml), hydrocortisone (10 nM), insulin (1 kg/ml), somatostatin (10 rig/ml), transferrin (5 kg/ml), TSH (1 mu/ml): this is referred to as 6H medium; a similar medium lacking TSH is referred to as 5H medium. of labeled oligosaccharide precursors: For the Incorporation experiments without TSH, the 6H medium was replaced with a 5H medium 48 h after plating, and the cells were used for experiment after 7-9 days; for the experiments with TSH, cells were plated 48 h before. At the time of experiment, the cells were incubated for 2 h in either 5H or 6H media containing one-fifth of the normal glucose concentration and for 5 h with either 50 pCi/ml of D-[2-3H] mannose or 50 pCi/ml of D-[1-3Hlgalactose. Purification and fragmentation of Tg: Tg was purified by a novel method, to be described elsewhere in detail. Subsequent steps included fractional precipitation in 1.4-1.8 M (NH4)2S04 and chromatography with Phenyl-sepharose CL4B (Pharmacia LKB), Biogel HTP (Bio-Rad), Sephacryl S-300 HR (Pharmacia LKB). The secreted Tg was fragmented with CNBr (10 mg/mg of Tg) in 70% (v/v) formic acid for 18 h at room temperature. Separation and identification of the fragments: the fragments were analyzed by reducing SDS-PAGE on 15-20% (w/v) total acrylamide gradient gels and autoradiography. For NH2-terminal peptide sequencing, samples of 0.5 mg of the digests were subjected to electrophoresis in the and transferred to PVDF presence of 100 mM sodium thioglycollate, membranes (Immobilon P, Millipore) by semi-dry blotting in 25 mM Tris base, 10 mM glycine; bands were stained briefly with Coomassie Brilliant Blue R-250, cut and subjected to gas-phase NHz-terminal microsequencing at the Protein Structure Laboratory, University of California at Davis (CA). 1625
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
RESULTS The Tg secreted by FRTL-5 cells was purified by a convenient method, to be described elsewhere, whereby it was freed of all contaminants (Fig. 1). Fig. 2 shows the CNBr fragments of the Tg secreted by FRTL-5 cells cultured with or without TSH, plus [3H]man or [3H]gal. Although equimolar amounts of Tg were loaded in the four lanes, better estimates of the relative labeling of different bands with each precursor will be obtained by internal comparison in each lane. In the absence of TSH, four bands were prominently labeled with [3H]man, with apparent masses of 41, 28, 26 and 18 kDa; the first three of these and a 34 kDa band were also lightly labeled with [sH]gal. The addition of TSH was associated with an increase of the [3H]man and a marked increase of the [3H]gal incorporation into the 26 kDa band, relative to the other bands. A slight increase of the [3H]gal incorporation characterized also a previously unnoticed 39 kDa band. The NH:!-terminal sequences of the 41, 28 and 26 kDa bands were obtained (Table 1) and aligned with the sequences deduced from the rTg gene [7] and the cDNAs of rTg [8], bTg [9] and hTg [lo] (Fig. 3). Predictions of their COOH-termini relay on the positions of methionines coming next in
TSH [3H]Man
+ +
-
+
+
PHIGal
-
45
-
30
-
20
-
14
1169468-
01
4530-
02
Fig. 1. Purification
of Tg secreted by FRTL-5 cells. Reducing SDSPAGE on a 10% total acrylamide gel stained with Coomassie Blue R-250. Lane I, ammonium sulphate precipitate of the whole cell culture medium; lane 2, purified Tg (2 Kg). The masses in kDa of the following standards are shown (from higher to lower): myosin, P-galactosidase, phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase. Fig. 2. Incorporation of labeled oligosaccharide precursors in the CNBr peptides of Tg secreted by FRTL-5 cells: effect of TSH. The autoradiograph is shown of a reducing 15-20% total acrylamide gradient gel. Cells were cultured with or without TSH and with D-[2-3Hlmannose or D-[ 1 -sH]galactose, as indicated at the top. The masses in kDa of the following standards are indicated (from higher to lower): ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme. 1626
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189, No. 3, 1992
Table
1. N Hz-terminal peptide sequences of the oligosaccharidelabeled CNBr peptides of Tg secreted by FRTL-5 cells
Apparent mass NW
NHz-terminal peptide sequence
41
GIQTXSEENl
28
GGSALSPAA
Residue numbers at extremes
Putative glycosylation sites
Calculated mass &Da)
229 - (508) (r) 2013 - (2292) (b)
30.5
4
614 - (796) (r) - (2578) (b)
21.0
2
14.0
3 (b)
2396
1 (r*)-
NIFEYQVD
26
(127)? (b,h)
1 X indicates a blank in the peptide sequence, corresponding codon in the rTg cDNA [8].
to a cystein
The peptide sequences were aligned with both the partial gene-derived (r*) [7] and cDNA-derived (r) [8] sequence of rTg and the full-length cDNAderived sequence of bTg (b) [9] and hTg (h) [lo]. Only the NH2 terminus of each peptide was determined, while the COOH terminus was inferred from the position of the next methionine. Apparent masses were observed in SDS-PAGE; calculated masses are based on the amino acid sequence only.
the
sequence.
peptides
The
agreed
apparent
well,
and predicted
considering
masses
the putative
sites
of
the
for
41
and
the addition
28 klIa of N-
mass of 2.0 kDa each [l]. The 26 kDa peptide corresponded to the NHz-terminus of Tg; its COOH-terminus was uncertain, since the rTg sequence in this region is unknown, whereas in bTg and hTg the first methionine occurs at position 127 [9, 101; indeed, a 26 kDa peptide, spontaneously cleaved from hTg,
linked
oligosaccharides,
ended
at residue
contributing
129 [ll];
an average
the slower
than
expected
migration,
for
such a
peptide length, could be due to a larger than average mass contribution of carbohydrates and to a high negative charge; complex, sulfate-containing oligosaccharide chains were described at residues 57 and 91 of hTg [12]. The 41 and 28 kDa peptides covered most of the COOH-terminal onefourth of Tg: the former started at residue 2013 (bTg numbering) and likely ended at methionine 2292, including four putative glycosylation sites (2104, 2151, 2232 and 2277); the latter started at residue 2396 and likely ended at methionine 2578, including two glycosylation sites (2426 and 2564); glycosylation was reported at residue 2562 of hTg [13]. Attempts at the sequencing of the 18 kDa band revealed multiple whose identification will require improved resolution. peptides, 1627
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COMMUNICATIONS
(r) (b)
Fig. 3. Localization of the oligosaccharide-labeled CNBr fragments of Tg within the gene- and cDNA-derived sequences of rat Tg. The gene-derived amino-terminal sequence of rTg (r*) [6] is shown in the uppermost line, followed by most of the partial cDNA-derived COOHterminal sequence of rTg [8]; residues in this part of Tg are numbered both as in the cDNA-derived sequence of rTg (r) [8] and in the aligned cDNA-derived sequence of bTg (b) [9]. Regions between two consecutive methionyl residues, likely corresponding to the fragments described in the text, are boxed; the residue numbers at their extremes are indicated; the NH2-termini were identified by peptide sequencing, and the COOH-termini were theoretically predicted. Putative sites of N-linked glycosylation are underlined; hormonogenic tyrosyl residues [14-181 are boxed.
DISCUSSION by FRTL-5 cells were identified. Two of them, with apparent masses of 41 and 28 kDa, covered a major portion of the COOH-terminal one-fourth of Tg and included all of its putative N-linked glycosylation sites. The high ratio of the [sHIman versus [sH]gal labeling of the 41 kDa band, notwithstanding the higher specific activity of the [sHIman preparation, seems to indicate the presence of high-mannose chains in this region of Tg. These likely prevailed in one or more of the peptides comigrating in the 18 kDa band. A peptide corresponding to the NHn-terminus of Tg was characterized by a distinct increase of the incorporation of [sHIman and, to a greater extent, of [sH]gal, under the TSH stimulation. The TSH-induced maturation Three
glycosylated
CNBr
peptides
of the Tg
1628
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the oligosaccharide chains of the Tg secreted by FRTL-5 cells, from the high-mannose to the complex type, was reported [6]. The present data show that this effect is mainly localized within a region where it may significantly influence the synthesis of thyroid hormones. Indeed, the 26 kDa peptide contained the main hormonogenic site of Tg, including the acceptor tyrosyl residue at position 5 [16-211 and the donor tyrosyl residue at position 130 [22]. It was reported that the stimulation by TSH is able to alter the priority by which the amino-terminal hormonogenic site is utilized in rabbit and guinea pig Tg, with respect to other hormonogenic sites located in other regions of Tg [17], and TSH-induced changes of the glycosylation were among the explanations hypotesized. The changes of the carbohydrate incorporation into the 26 kDa peptide likely reflect an increase of the N-linked glycosylation at the NHs-terminus of Tg, with the addition of high-mannose oligosaccharide chains and, to a greater extent, the maturation of existing high-mannose chains to complex ones; they may also reflect the addition or the elongation of O-linked oligosaccharide chains, provided that any of those described in hTg occurs in rat Tg, and that any [sH]galactose was converted to (N-acetyl-)[3H]galactosamine. The precise localization and structural characterization of the oligosaccharide chains involved are the aim of further work. ACKNOWLEDGMENTS The support of Prof. Gaetano Salvatore, Eduardo Consiglio, Giuseppe Palumbo is gratefully acknowledged. This work was partly supported by the Progetto Finaiizzato Biotecnologie e Strumentazione, C.N.R., Italy. REFERENCES 1. Arima, T., Spiro, M .J., and Spiro, R.G. (1972) J. Biol.Chem. 247, 18251835. 2. Spiro, R. G., and Bhoyroo, V. D. (1988) J. Biol.Chem. 263, 14351-14358. 3. Spiro, M .J. (1977) J. BioLChem. 252, 5424-5430. 4. Schneider, A. B., McCurdy, A., Chang, T., Dudlak, D., and Magner, J. (1988) Endocrinology 122, 2428-2435. 5. Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293. 6. Di Jeso, B., Liguoro, D., Ferranti, P., Marinaccio, M ., Acquaviva, R., Formisano, S., and Consiglio, E. (1992) J. Biol. Chem. 267, 1983-1944. 7. Musti, A. M ., Avvedimento, V. E., Polistina, C., Ursini, V. M ., Obici, S., Nitsch, L., Cocozza, S., and Di Lauro, R. (1986) Proc. Nutl. Acad. Sci. USA 83, 323-327. 8. Di Lauro, R., Obici, S., Condliffe, D., Ursini, V. M ., Musti, A. M ., Moscatelli, C., and Avvedimento, V. E. (1985) Eur. J. Biochem. 148, 7-11. 9. Mercken, L., Simons, M .-J., Swillens, S., Massaer, M ., and Vassar& G. (1985) Nature 316, 647-651. 1629
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10. Malthiery, Y., and Lissitzky, S. (1987) Eur. J. Biochem. 165, 491-498. 11. Marriq, C., Lejeune, P.-J., Venot, N., and Vinet, L. (1989) FEBS Lett. 242, 414-418. 12. Franc, J.-L., Venot, N., and Marriq, C. (1990) Biochem. Biophys. Res. Comm. 166, 937-944. 13. Rawitch, A. B., Lias, T. H., and Pierce, J. G. (1968) Biochim. Biophys. Acta 160, 360-367. 14. Marriq, C., Rolland, M ., and Lissitzky, S. (1983) Biochem. Biophys. Res. Comm. 112, 206-213. 15. Marriq, C., Rolland, M ., and Lissitzky, S. (1982) EMBO J. 1, 397-401. 16. Dunn, J. T., Anderson, P. C., Fox, J. W., Fassler, C. A., Dunn, A. D., Hite, L. A., and Moore, R. C. (1987) J. Biol. Chem. 262, 16948-16952. 17. Fassler, C. A., Dunn, J. T., Anderson, P. C., Fox, J. W., Dunn, A. D., Hite, L. A., Moore, R. C., and Kim, P. S. (1988) J. Biol. Chem. 263, 17366-17371. 18. Lamas, L., Anderson, P. C., Fox, J. W., and Dunn, J. T. (1989) J. Biol. Chem. 264, 13541-13545. 19. Rawitch, A. B., Chemoff, S. B., Litwer, M . R., Rouse, J. B., and Hamilton, J. W. (1983) J. Biol. Chem. 258, 2079-2082. 20. Lejeune, P.-J., Marriq, C., Rolland, M ., and Lissitzky, S. (1983) Biochem. Biophys. Res. Comm. 114, 73-80. 21. Rawitch, A. B., Litwer, M . R., Gregg, J., Dziadik-Turner, C., Rouse, J. B., and Hamilton, J.W. (1984) Biochem. Biophys. Res. Comm. 118, 423-429. 22. Marriq, C., Lejeune, P.-J., Venot, N., and Vinet, L. (1991) Mel Cell. Endocrinol. 81, 155-164.
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Vol. 189, No. 3, 1992 December 30, 1992
TSH-INDUCED GALACTOSE INCORPORATION AT THE NH2 TERMINUS OF THYROGLOBULIN SECRETED BY FRTL-5 CELLS Bruno Di Jesol and Fabrizio Gentile Centro di Endocrinologia e Oncologia Sperimentale de1 C.N.R. and Dipartimento di Biologia e Patologia Cellulare e Molecolare, University of Naples 2nd Medical School, Via Pansini, 5, 80131 Naples, Italy Received
November
20,
1992
SUMMARY : FRTL-5 cells were cultured in media containing standard growth factors with or without TSH, plus labeled precursors of N-linked secreted in the medium was oligosaccharide chains. The thyroglobulin purified and fragmented with CNBr. Three peptides were identified by NH2-terminal sequencing, that were labeled mainly with D-[2-3H]mannose, independent of TSH. One of them, corresponding to the NHz-terminus of thyroglobulin, incorporated both more D-[2-3Hlmannose and more D-[ l3Hlgalactose upon TSH addition. These data likely reflect a TSH-induced increment of N-linked glycosylation at the NH2-terminus of thyroglobulin, 0 1992 mostly with the maturation of high-mannose to complex chains. Rcademlc mes*,Inc.
Thyroglobulin is the dimeric glycoprotein of 660 kDa providing the backbone for the synthesis of thyroid hormones. Carbohydrates compose 10% of its mass; major oligosaccharide chains of human and bovine Tg include N-linked chains of high-mannose type, consisting of mannose and and complex type, containing in addition galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid and sulfate groups [ 1, 21. O-linked chains of human Tg include galactosamine-containing chains and chondroitin 6sulfate-like chains, containing N-acetylgalactosamine and glucuronic acid
1 To whom correspondence should be addressed. Abbreviations: Tg, thyroglobulin; rTg, rat thyroglobulin; bTg, bovine hTg, human thyroglobulin; [sHIman, D-[2-3H] mannose; thyroglobulin; [3H]gal, D-[ 1-sH]galactose; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 0006-291X/92
$4.00
Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1624
Vol.
189,
No.
3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
[2-41. TSH promotes the sialylation of the oligosaccharide chains of Tg secreted by pig thyroid cells in primary culture [5] and the maturation of the high-mannose to complex chains of the Tg secreted by FRTL-5 cells [6]. This study was undertaken in order to determine: 1) the kinds of Nlinked oligosaccharide chains present in defined regions of the sequence of Tg secreted by FRTL-5 cells; 2) the location of the oligosaccharide chains maturing from the high-mannose to the complex type under the effect of TSH. The Tg secreted by FRTL-5 cells, cultured both in the absence and in the presence of TSH, plus labeled precursors of oligosaccharide chains (D[2-JH]mannose for high-mannose chains and D-[l-3Hlgalactose for complex chains), was purified and fragmented with CNBr. The peptides produced were isolated and identified by NHz-terminal sequencing and alignment with the gene- and cDNA-derived sequences of rTg, bTg and hTg [7-101. MATERIALS
AND METHODS
Materials: D-[2-3Hlmannose (10 Ci/mmol) and D-[ 1-3Hlgalactose (5 Ci/mmol) from Amersham, hormones from Calbiochem (Insulin) or Sigma. Cell culture: FRTL-5 cells (ATCC CRL 8305) were routinely cultured in Coon’s modified Ham’s F12 medium plus 5% calf serum, glycyl-histidyllysine (10 rig/ml), hydrocortisone (10 nM), insulin (1 kg/ml), somatostatin (10 rig/ml), transferrin (5 kg/ml), TSH (1 mu/ml): this is referred to as 6H medium; a similar medium lacking TSH is referred to as 5H medium. of labeled oligosaccharide precursors: For the Incorporation experiments without TSH, the 6H medium was replaced with a 5H medium 48 h after plating, and the cells were used for experiment after 7-9 days; for the experiments with TSH, cells were plated 48 h before. At the time of experiment, the cells were incubated for 2 h in either 5H or 6H media containing one-fifth of the normal glucose concentration and for 5 h with either 50 pCi/ml of D-[2-3H] mannose or 50 pCi/ml of D-[1-3Hlgalactose. Purification and fragmentation of Tg: Tg was purified by a novel method, to be described elsewhere in detail. Subsequent steps included fractional precipitation in 1.4-1.8 M (NH4)2S04 and chromatography with Phenyl-sepharose CL4B (Pharmacia LKB), Biogel HTP (Bio-Rad), Sephacryl S-300 HR (Pharmacia LKB). The secreted Tg was fragmented with CNBr (10 mg/mg of Tg) in 70% (v/v) formic acid for 18 h at room temperature. Separation and identification of the fragments: the fragments were analyzed by reducing SDS-PAGE on 15-20% (w/v) total acrylamide gradient gels and autoradiography. For NH2-terminal peptide sequencing, samples of 0.5 mg of the digests were subjected to electrophoresis in the and transferred to PVDF presence of 100 mM sodium thioglycollate, membranes (Immobilon P, Millipore) by semi-dry blotting in 25 mM Tris base, 10 mM glycine; bands were stained briefly with Coomassie Brilliant Blue R-250, cut and subjected to gas-phase NHz-terminal microsequencing at the Protein Structure Laboratory, University of California at Davis (CA). 1625
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 189, No. 3, 1992
RESULTS The Tg secreted by FRTL-5 cells was purified by a convenient method, to be described elsewhere, whereby it was freed of all contaminants (Fig. 1). Fig. 2 shows the CNBr fragments of the Tg secreted by FRTL-5 cells cultured with or without TSH, plus [3H]man or [3H]gal. Although equimolar amounts of Tg were loaded in the four lanes, better estimates of the relative labeling of different bands with each precursor will be obtained by internal comparison in each lane. In the absence of TSH, four bands were prominently labeled with [3H]man, with apparent masses of 41, 28, 26 and 18 kDa; the first three of these and a 34 kDa band were also lightly labeled with [sH]gal. The addition of TSH was associated with an increase of the [3H]man and a marked increase of the [3H]gal incorporation into the 26 kDa band, relative to the other bands. A slight increase of the [3H]gal incorporation characterized also a previously unnoticed 39 kDa band. The NH:!-terminal sequences of the 41, 28 and 26 kDa bands were obtained (Table 1) and aligned with the sequences deduced from the rTg gene [7] and the cDNAs of rTg [8], bTg [9] and hTg [lo] (Fig. 3). Predictions of their COOH-termini relay on the positions of methionines coming next in
TSH [3H]Man
+ +
-
+
+
PHIGal
-
45
-
30
-
20
-
14
1169468-
01
4530-
02
Fig. 1. Purification
of Tg secreted by FRTL-5 cells. Reducing SDSPAGE on a 10% total acrylamide gel stained with Coomassie Blue R-250. Lane I, ammonium sulphate precipitate of the whole cell culture medium; lane 2, purified Tg (2 Kg). The masses in kDa of the following standards are shown (from higher to lower): myosin, P-galactosidase, phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase. Fig. 2. Incorporation of labeled oligosaccharide precursors in the CNBr peptides of Tg secreted by FRTL-5 cells: effect of TSH. The autoradiograph is shown of a reducing 15-20% total acrylamide gradient gel. Cells were cultured with or without TSH and with D-[2-3Hlmannose or D-[ 1 -sH]galactose, as indicated at the top. The masses in kDa of the following standards are indicated (from higher to lower): ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme. 1626
Vol.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
189, No. 3, 1992
Table
1. N Hz-terminal peptide sequences of the oligosaccharidelabeled CNBr peptides of Tg secreted by FRTL-5 cells
Apparent mass NW
NHz-terminal peptide sequence
41
GIQTXSEENl
28
GGSALSPAA
Residue numbers at extremes
Putative glycosylation sites
Calculated mass &Da)
229 - (508) (r) 2013 - (2292) (b)
30.5
4
614 - (796) (r) - (2578) (b)
21.0
2
14.0
3 (b)
2396
1 (r*)-
NIFEYQVD
26
(127)? (b,h)
1 X indicates a blank in the peptide sequence, corresponding codon in the rTg cDNA [8].
to a cystein
The peptide sequences were aligned with both the partial gene-derived (r*) [7] and cDNA-derived (r) [8] sequence of rTg and the full-length cDNAderived sequence of bTg (b) [9] and hTg (h) [lo]. Only the NH2 terminus of each peptide was determined, while the COOH terminus was inferred from the position of the next methionine. Apparent masses were observed in SDS-PAGE; calculated masses are based on the amino acid sequence only.
the
sequence.
peptides
The
agreed
apparent
well,
and predicted
considering
masses
the putative
sites
of
the
for
41
and
the addition
28 klIa of N-
mass of 2.0 kDa each [l]. The 26 kDa peptide corresponded to the NHz-terminus of Tg; its COOH-terminus was uncertain, since the rTg sequence in this region is unknown, whereas in bTg and hTg the first methionine occurs at position 127 [9, 101; indeed, a 26 kDa peptide, spontaneously cleaved from hTg,
linked
oligosaccharides,
ended
at residue
contributing
129 [ll];
an average
the slower
than
expected
migration,
for
such a
peptide length, could be due to a larger than average mass contribution of carbohydrates and to a high negative charge; complex, sulfate-containing oligosaccharide chains were described at residues 57 and 91 of hTg [12]. The 41 and 28 kDa peptides covered most of the COOH-terminal onefourth of Tg: the former started at residue 2013 (bTg numbering) and likely ended at methionine 2292, including four putative glycosylation sites (2104, 2151, 2232 and 2277); the latter started at residue 2396 and likely ended at methionine 2578, including two glycosylation sites (2426 and 2564); glycosylation was reported at residue 2562 of hTg [13]. Attempts at the sequencing of the 18 kDa band revealed multiple whose identification will require improved resolution. peptides, 1627
Vol.
189,
No.
BIOCHEMICAL
3, 1992
“PK.5
FISK
967 2750
AND BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(r) (b)
Fig. 3. Localization of the oligosaccharide-labeled CNBr fragments of Tg within the gene- and cDNA-derived sequences of rat Tg. The gene-derived amino-terminal sequence of rTg (r*) [6] is shown in the uppermost line, followed by most of the partial cDNA-derived COOHterminal sequence of rTg [8]; residues in this part of Tg are numbered both as in the cDNA-derived sequence of rTg (r) [8] and in the aligned cDNA-derived sequence of bTg (b) [9]. Regions between two consecutive methionyl residues, likely corresponding to the fragments described in the text, are boxed; the residue numbers at their extremes are indicated; the NH2-termini were identified by peptide sequencing, and the COOH-termini were theoretically predicted. Putative sites of N-linked glycosylation are underlined; hormonogenic tyrosyl residues [14-181 are boxed.
DISCUSSION by FRTL-5 cells were identified. Two of them, with apparent masses of 41 and 28 kDa, covered a major portion of the COOH-terminal one-fourth of Tg and included all of its putative N-linked glycosylation sites. The high ratio of the [sHIman versus [sH]gal labeling of the 41 kDa band, notwithstanding the higher specific activity of the [sHIman preparation, seems to indicate the presence of high-mannose chains in this region of Tg. These likely prevailed in one or more of the peptides comigrating in the 18 kDa band. A peptide corresponding to the NHn-terminus of Tg was characterized by a distinct increase of the incorporation of [sHIman and, to a greater extent, of [sH]gal, under the TSH stimulation. The TSH-induced maturation Three
glycosylated
CNBr
peptides
of the Tg
1628
secreted
Vol.
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of the oligosaccharide chains of the Tg secreted by FRTL-5 cells, from the high-mannose to the complex type, was reported [6]. The present data show that this effect is mainly localized within a region where it may significantly influence the synthesis of thyroid hormones. Indeed, the 26 kDa peptide contained the main hormonogenic site of Tg, including the acceptor tyrosyl residue at position 5 [16-211 and the donor tyrosyl residue at position 130 [22]. It was reported that the stimulation by TSH is able to alter the priority by which the amino-terminal hormonogenic site is utilized in rabbit and guinea pig Tg, with respect to other hormonogenic sites located in other regions of Tg [17], and TSH-induced changes of the glycosylation were among the explanations hypotesized. The changes of the carbohydrate incorporation into the 26 kDa peptide likely reflect an increase of the N-linked glycosylation at the NHs-terminus of Tg, with the addition of high-mannose oligosaccharide chains and, to a greater extent, the maturation of existing high-mannose chains to complex ones; they may also reflect the addition or the elongation of O-linked oligosaccharide chains, provided that any of those described in hTg occurs in rat Tg, and that any [sH]galactose was converted to (N-acetyl-)[3H]galactosamine. The precise localization and structural characterization of the oligosaccharide chains involved are the aim of further work. ACKNOWLEDGMENTS The support of Prof. Gaetano Salvatore, Eduardo Consiglio, Giuseppe Palumbo is gratefully acknowledged. This work was partly supported by the Progetto Finaiizzato Biotecnologie e Strumentazione, C.N.R., Italy. REFERENCES 1. Arima, T., Spiro, M .J., and Spiro, R.G. (1972) J. Biol.Chem. 247, 18251835. 2. Spiro, R. G., and Bhoyroo, V. D. (1988) J. Biol.Chem. 263, 14351-14358. 3. Spiro, M .J. (1977) J. BioLChem. 252, 5424-5430. 4. Schneider, A. B., McCurdy, A., Chang, T., Dudlak, D., and Magner, J. (1988) Endocrinology 122, 2428-2435. 5. Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293. 6. Di Jeso, B., Liguoro, D., Ferranti, P., Marinaccio, M ., Acquaviva, R., Formisano, S., and Consiglio, E. (1992) J. Biol. Chem. 267, 1983-1944. 7. Musti, A. M ., Avvedimento, V. E., Polistina, C., Ursini, V. M ., Obici, S., Nitsch, L., Cocozza, S., and Di Lauro, R. (1986) Proc. Nutl. Acad. Sci. USA 83, 323-327. 8. Di Lauro, R., Obici, S., Condliffe, D., Ursini, V. M ., Musti, A. M ., Moscatelli, C., and Avvedimento, V. E. (1985) Eur. J. Biochem. 148, 7-11. 9. Mercken, L., Simons, M .-J., Swillens, S., Massaer, M ., and Vassar& G. (1985) Nature 316, 647-651. 1629
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10. Malthiery, Y., and Lissitzky, S. (1987) Eur. J. Biochem. 165, 491-498. 11. Marriq, C., Lejeune, P.-J., Venot, N., and Vinet, L. (1989) FEBS Lett. 242, 414-418. 12. Franc, J.-L., Venot, N., and Marriq, C. (1990) Biochem. Biophys. Res. Comm. 166, 937-944. 13. Rawitch, A. B., Lias, T. H., and Pierce, J. G. (1968) Biochim. Biophys. Acta 160, 360-367. 14. Marriq, C., Rolland, M ., and Lissitzky, S. (1983) Biochem. Biophys. Res. Comm. 112, 206-213. 15. Marriq, C., Rolland, M ., and Lissitzky, S. (1982) EMBO J. 1, 397-401. 16. Dunn, J. T., Anderson, P. C., Fox, J. W., Fassler, C. A., Dunn, A. D., Hite, L. A., and Moore, R. C. (1987) J. Biol. Chem. 262, 16948-16952. 17. Fassler, C. A., Dunn, J. T., Anderson, P. C., Fox, J. W., Dunn, A. D., Hite, L. A., Moore, R. C., and Kim, P. S. (1988) J. Biol. Chem. 263, 17366-17371. 18. Lamas, L., Anderson, P. C., Fox, J. W., and Dunn, J. T. (1989) J. Biol. Chem. 264, 13541-13545. 19. Rawitch, A. B., Chemoff, S. B., Litwer, M . R., Rouse, J. B., and Hamilton, J. W. (1983) J. Biol. Chem. 258, 2079-2082. 20. Lejeune, P.-J., Marriq, C., Rolland, M ., and Lissitzky, S. (1983) Biochem. Biophys. Res. Comm. 114, 73-80. 21. Rawitch, A. B., Litwer, M . R., Gregg, J., Dziadik-Turner, C., Rouse, J. B., and Hamilton, J.W. (1984) Biochem. Biophys. Res. Comm. 118, 423-429. 22. Marriq, C., Lejeune, P.-J., Venot, N., and Vinet, L. (1991) Mel Cell. Endocrinol. 81, 155-164.
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